Chroman derivatives, medicaments and use in therapy

ABSTRACT

Novel chroman derivatives and intermediate compounds, compositions containing same, methods for their preparation and uses thereof as therapeutic agents particularly as anti-cancer and chemotherapeutic selective agents are described.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.13/891,975, filed May 10, 2013, now U.S. Pat. No. 8,957,109, issued Feb.17, 2015, which is a continuation of U.S. application Ser. No.13/415,697, filed Mar. 8, 2012, now U.S. Pat. No. 8,461,361, issued Jun.11, 2013, which is a continuation of U.S. application Ser. No.13/231,794, filed Sep. 13, 2011, now U.S. Pat. No. 8,163,795, issuedApr. 24, 2012, which is a divisional patent application of U.S.application Ser. No. 11/230,505, filed Sep. 21, 2005 entitled “CHROMANDERIVATIVES, MEDICAMENTS AND USE IN THERAPY”, now U.S. Pat. No.8,080,675, issued Dec. 20, 2011, which claims benefit of U.S.Provisional Application No. 60/611,299, entitled “COMPOUNDS” filed onSep. 21, 2004; of U.S. Provisional Application No. 60/676,934, entitled“CHROMAN DERIVED COMPOUNDS & FORMULATIONS THEREOF FOR USE IN THERAPY”filed on May 3, 2005; of PCT application No. PCT/AU04/01619, entitled“COMBINATIONAL RADIOTHERAPY AND CHEMOTHERAPY COMPOSITIONS AND METHODS”filed on Nov. 19, 2004; of AU application 2004/906363, entitled“COMPOUNDS” filed Nov. 5, 2004; and of JP application 2004/315009entitled “COMPOUNDS” filed Oct. 29, 2004; all of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to certain novel chroman derivatives,compositions containing same, methods for their preparation and usesthereof as therapeutic agents particularly as anti-cancer andchemotherapeutic selective agents.

BACKGROUND OF THE INVENTION

Over 700 different naturally occurring isoflavones are known some ofwhich have biological properties with potential therapeutic benefit.

U.S. Pat. No. 5,726,202 generically discloses certain isoflavancompounds, particularly 3,4-diarylchroman and centchroman for thetreatment of benign prostatic hypertrophy.

WO 01/17986 also discloses certain isoflavan compounds.

SUMMARY OF THE INVENTION

Surprisingly, the present inventors have found a novel group ofcompounds of the general formula (I) which exhibit important therapeuticactivities including strong anti-cancer activity, chemotherapeuticselectivity and radiosensitisation of cancers.

Thus according to an aspect of the present invention there is provided acompound of the general formula (I):

wherein

-   R₁ is hydrogen, alkyl, cycloalkyl or C(O)R₇,-   R₂ and R₃ are independently hydrogen, hydroxy, alkoxy, alkyl,    cycloalkyl, halo or OC(O)R₇, with the exception that R₂ and R₃ are    not both hydrogen,-   R₄, R₅ and R₆ are independently hydrogen, hydroxy, alkoxy, alkyl,    cycloalkyl, acyl, amino, C₁₋₄-alkylamino or di(C₁₋₄-alkyl)amino,    OC(O)R₇ or OR₈,-   R₇ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino, and-   R₈ is aryl such as phenyl or arylalkyl such as benzyl, and-   R₉ is hydrogen hydroxy, alkyl, alkoxy, cycloalkyl or halo, or a    pharmaceutically acceptable salt or derivative thereof.

In a preferred embodiment of the present invention R₉ is hydrogen.Accordingly, in another aspect of the invention there is provided acompound of the formula (I-a):

wherein

-   R₁ is hydrogen, alkyl, cycloalkyl or C(O)R₇,-   R₂ and R₃ are independently hydrogen, hydroxy, alkoxy, halo or    OC(O)R₇, with the exception that R₂ and R₃ are not both hydrogen,-   R₄, R₅ and R₆ are independently hydrogen, hydroxy, alkoxy, alkyl,    cycloalkyl, acyl, OC(O)R₇, amino, and-   R₇ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino.

According to another aspect of the present invention there is provided aprocess for the preparation of a compound of formula (I) comprising thestep of reacting the keto group of a compound of the formula (II):

or the analogue thereof including a substituent which corresponds to R₉in compounds of formula (I) wherein

-   R₁, is alkyl or a protecting group such as Si(R₁₀)₃,-   R₂ and R₃ are independently hydrogen, alkoxy or OSi(R₁₀)₃, with the    exception that R₂ and R₃ are not both hydrogen, and-   R₁₀ is independently alkyl or aryl,    with an arylating agent W⁻M⁺,    wherein-   W⁻ is an optionally substituted aryl radical, and-   M⁺ is one or more counter ions, preferably [MgBr]⁺,    to form the intermediate tertiary alcohol of formula (III):

or protected derivative thereof or a salt thereof (or an analoguethereof including a substituent which corresponds to R₉ in compounds offormula (I)) and which is dehydrated to form a compound of formula (IV):

(or an analogue thereof including a substituent which corresponds to R₉in compounds of formula (I)) the double bond of which is subsequentlyreduced, for example, by hydrogenation and optionally deprotected toform a compound of formula (I).

According to another aspect of the present invention there is provided acompound of the general formula (III), compositions containing same anduses thereof.

In another aspect, there is provided a compound of the general formula(IV), compositions containing same and uses thereof.

Thus, according to another aspect of the present invention there isprovided the use of a compound of formula (I) in therapy, particularlychemotherapy and/or as a radiosensitising or chemosensitising agent.

According to another aspect of the present invention there is provided amethod for the treatment, prevention or amelioration of a disease ordisorder, which comprises administering to a subject one or morecompounds of the formula (I) or a pharmaceutically acceptable salt orderivative thereof optionally in association with a carrier and/orexcipient.

According to another aspect of the present invention there is providedthe use of one or more compounds of formula (I) or a pharmaceuticallyacceptable salt or derivative thereof in the manufacture of a medicamentfor the treatment of a disease or disorder.

According to another aspect of the present invention there is providedan agent for the treatment, prophylaxis or amelioration of a disease ordisorder which agent comprises one or more compounds of formula (I) or apharmaceutically acceptable salt or derivative thereof.

According to another aspect of the present invention there is provided apharmaceutical composition which comprises one or more compounds offormula (I) or a pharmaceutically acceptable salt or derivative thereofin association with one or more pharmaceutical carriers, excipients,auxiliaries and/or diluents.

According to another aspect of the present invention there is provided adrink or food-stuff, which contains one or more compounds of formula (I)or a pharmaceutically acceptable salt or derivative thereof.

These and other aspects of the invention will become evident from thedescription and claims which follow, together with the accompanyingdrawings.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 represents a comparison of dehydroequol (DHE graph A),3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (HMC compound 1according to the invention graph B) and cisplatin (graph C) toxicity inneonatal foreskin fibroblasts.

FIG. 2 represents HMC efficacy in melanoma cells in comparison withcisplatin.

FIG. 3 represents a pharmacokinetic profile of free and total forms ofHMC (A) and DHE (B) after p.o (peri oral) administration to BALB/c mice(50 mg/kg).

FIG. 4 represents a comparison of the pharmacokinetic profile the HMCconcentration in serum after i.v (intravenously) and i.p(intraperitoneally) administration of HMC formulated in 20%hydroxypropyl-beta-cyclodextrin at a dose of 50 mg/kg.

FIG. 5 represents comparative mean tumour volume data taken from nudemice bearing HPAC pancreatic cancer tumours treated with either i.pdosed 20% HPBCD (vehicle control, qdx15) or HMC (100 mg/kg, qdx15). Datarepresented as mean±SEM*, student's T-test, p<0.01.

FIG. 6 represents comparative mean terminal tumour mass data taken fromnude mice bearing HPAC pancreatic cancer tumours treated with either i.pdosed 20% HPBCD (vehicle control, qdx15) or HMC (100 mg/kg, qdx15). Datarepresented as mean±SEM*, student's T-test, p<0.01.

FIG. 7 represents comparative mean terminal tumour mass data taken fromnude mice bearing HPAC pancreatic cancer tumours treated with either i.pdosed 20% HPBCD (vehicle control, qdx15) or HMC (100 mg/kg, qdx15). Datarepresented as mean±SEM*, student's T-test, p<0.01.

FIG. 8 represents a summary of apoptosis incidence in DHE and HMCtreated melanoma cells over a 24 and 48 hour period.

FIG. 9 represents selective initiation of programmed cell death in HMCand DHE treated malignant melanoma cells (Mel-RM and Me4405). The sameconcentration of DHE and HMC and exposure times do not induce apoptosisin normal fibroblasts (MRC-5).

FIG. 10A and FIG. 10B represent a 3D analysis of HMC-cisplatin synergycytotoxicity data in the MM200 melanoma cell line. HMC-cisplatincombinations were assessed using a 5-day combination protocol (FIG.10A), or a 24 hr HMC→anti-cancer sequence (FIG. 10B). For eachcombination experiment HMC was assessed at 10, 5, 2 and 1 μM. See Table8 for raw data.

FIG. 11 represents the percentage inhibition of TNFα in murinemacrophages by compounds 6 and 7 of the invention.

FIG. 12 represents the ¹H n.m.r. spectrum of3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that a class of isoflavan derivativesof the general formula (I) show surprising and unexpected biological andpharmaceutical properties.

The compounds of formula (I) of the invention are believed to havefavourable toxicity profiles with normal cells and good bioavailability.Surprisingly the compounds of the invention exhibit anti-canceractivity, significantly better than or at least comparable to knowncancer treatments.

The compounds of formula (I) are cytostatic and cytotoxic against abroad range of cancer cells of human and animal origin. By cancer cells,it is meant cells that display malignant characteristics and which aredistinguished from non-cancer cells by unregulated growth and behaviourwhich usually ultimately is life-threatening unless successfullytreated.

The cancer cells that have been found to be responsive to compounds offormula (I) are of epithelial origin (for example, prostate, ovarian,cervical, breast, gall-bladder, pancreatic, colorectal, renal, andnon-small lung cancer cells), of mesenchymal origin (for example,melanoma, mesothelioma and sarcoma cancer cells), and of neural origin(for example glioma cancer cells). It is highly unusual and surprisingto find a related group of compounds that display such potentcytotoxicity against cancer cells, but with low toxicity againstnon-cancer cells such as keratinocytes derived from human foreskin. Suchcancer cell selectivity is highly unusual and unexpected.

Advantageously the compounds of formula (I) show cytotoxicity againstcancer cells that are well recognised for being poorly sensitive tostandard anti-cancer drugs. It is highly unusual and unexpected to findsuch potent activity against cancers, for example, cholangiocarcinoma,pancreatic adenocarcinoma and melanoma.

Advantageously the compounds of formula (I) also unexpectedly display anability to radio-sensitise cancer cells, by which it is meant that thesecompounds either lower the amount of gamma-irradiation that is requiredto kill the cells, or they convert cancer cells from a state ofradio-resistance to a radio-sensitive state.

Additionally the compounds of formula (I) are thought to possesschemo-sensitising activity, that is they increase the cytotoxicity ofchemotherapeutic agents, especially to cancer cells, and/or convertcancerous cells from a state of chemo-resistance to a chemo-sensitivestate.

Compounds of the invention may also provide chemo and/orradio-protective properties for non-cancerous cells. This hassignificant therapeutic implications because the traumatic side-effectsof chemotherapy and radiotherapy are caused by the toxicity of thetraditional treatments to non-cancerous cells.

The properties described above offer significant clinical advantages.

The radio and/or chemo-protective properties of the compounds of theinvention may be employed to protect healthy individuals from theeffects of radiation and/or chemical toxins, or lessen the effects ofthe same.

Thus, the invention also provides the use of compounds of formula (I) totreat patients with cancer by either reducing the rate of growth of suchtumours or by reducing the size of such tumours through therapy withsaid compounds alone, and/or in combination with each other, and/or incombination with other anti-cancer agents, and/or in combination withradiotherapy.

The use of compounds of the present invention either alone or incombination therapy as described above may reduce the adverseside-effects often experienced by patients when treated with standardanti-cancer treatments. The use of compounds of the invention may meanthat lower doses can be employed in such therapy which represents animportant advance for cancer sufferers.

Preferably R₃ in compounds of formula (I) is in the 3-position.

In another aspect of the invention R₉ is C₁₋₄-alkyl, such as methyl.

Preferably in compounds of formula (I-a):

-   R₁ is hydrogen, C₁₋₄-alkyl or C(O)R₇,-   R₂ and R₃ are independently hydrogen, hydroxy, C₁₋₄-alkoxy, halo or    OC(O)R₇, provided that R₂ and R₃ are not both hydrogen,-   R.₄, R₅ and R₆ are independently hydrogen, hydroxy, alkoxy, alkyl,    cycloalkyl, acyl, OC(O)R₇, and-   R₇ is C₁₋₄-alkyl, phenyl or benzyl,    or a pharmaceutically acceptable salt or derivative thereof.

More preferably in compounds of formula (I-a):

-   R₁ is hydrogen, methyl, ethyl, propyl, isopropyl or acetyl,-   R₂ and R₃ are independently hydrogen, hydroxy, methoxy, ethoxy,    propoxy, isopropoxy, bromo, chloro, fluoro or acetyloxy, with the    exception that R₂ and R₃ are not both hydrogen,-   R₄ is hydrogen, hydroxy, methoxy, ethoxy, propoxy, isopropoxy or    acetyloxy, and-   R₅ and R₆ are independently hydrogen, hydroxy, methoxy, ethoxy,    propoxy, isopropoxy, acetyl, or acetyloxy,    or a pharmaceutically acceptable salt or derivative thereof.

Particular preferred compounds of formula (I-a) have the followingsubstituents where:

-   R₁ is hydrogen, methyl or acetyl,-   R₂ and R₃ are independently hydrogen, hydroxy, methoxy, bromo or    acetyloxy, with the exception that R₂ and R₃ are not both hydrogen,-   R₄ and R₆ are independently hydrogen, hydroxy, methoxy or acetyloxy,    and-   R₅ is hydrogen,    or a pharmaceutically acceptable salt or derivative thereof.

The invention also extends to compounds of formula (I-b):

wherein:

-   R₁ represents hydrogen or C₁₋₆-alkyl, more preferably hydrogen or    methyl, especially hydrogen.-   R2 represents hydrogen, hydroxy or C₁₋₆-alkoxy such as methoxy,    ethoxy, propoxy, more preferably hydroxy or methoxy, especially    hydroxy.-   R₃ represents hydrogen, hydroxy, C₁₋₆-alkoxy such as methoxy,    ethoxy, propoxy, more preferably hydrogen or methoxy, especially    hydrogen, with the proviso that R₂ and R₃ do not both represent    hydrogen,-   R₄ represents hydrogen, hydroxy, C₁₋₆-alkoxy such as methoxy,    ethoxy, propoxy, C₁₋₆-alkyl such as methyl, ethyl, propyl,    isopropyl, especially hydrogen, hydroxy, methoxy or methyl    particularly methoxy or hydroxy,-   R₅ represents hydrogen, C₁₋₆-alkoxy, especially hydrogen, methoxy,    hydroxy, particularly hydrogen,    or a pharmaceutically acceptable salt or derivative thereof.

Preferred compounds of the invention include those of the generalformula (I-c):

wherein:

-   R₁ is hydrogen or C₁-C₆ alkyl such as methyl, ethyl, propyl,    isopropyl, butyl, isobutyl, secbutyl, tertiary butyl,-   R₂ is hydroxy or C₁₋₆-alkoxy such as methoxy, ethoxy, propoxy,    isopropoxy, butoxy, isobutoxy, secbutoxy, tertiary butoxy, and-   R₄ is hydroxy or C₁₋₆-alkoxy such as methoxy, ethoxy, propoxy,    isopropoxy, butoxy, isobutoxy, secbutoxy, tertiary butoxy    or a pharmaceutically acceptable salt or a derivative thereof.

More preferably in compounds of formula (I-c) R₁ is hydrogen or methyl,especially hydrogen.

More preferably in compounds of formula (I-c) R₂ is hydroxy or methoxy,especially hydroxy.

More preferably in compounds of formula (I-c) R₄ is hydroxy or methoxy,especially methoxy.

In an alternative aspect the invention provides compounds of formula(I-d):

wherein:

-   R₁ is hydrogen, alkyl, cycloalkyl or C(O)R₇, and-   R₃ is hydroxy, alkoxy, alkyl, cycloalkyl, halo or OC(O)R₇, with the    exception that R₂ and R₃ are not both hydrogen,-   R₄ is hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, amino,    C₁₋₄-alkylamino or di(C₁₋₄-alkyl)amino or OC(O)R₇, and-   R₇ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino.

In a further alternative aspect the invention provides compounds offormula (I-e):

wherein:

-   R₁ is hydrogen, alkyl, cycloalkyl or C(O)R₇, and-   R₂ and R₃ are independently hydrogen, hydroxy, alkoxy, alkyl,    cycloalkyl, halo or OC(O)R₇, with the exception that R₂ and R₃ are    not both hydrogen,

Preferably in compounds of formula (I-e) R₁ represents hydrogen ormethyl, especially hydrogen.

Preferably in compounds of formula (I-e) R₂ represents hydroxy or C₁-C₆alkoxy such as methoxy.

In compounds of formula (I-e) preferably R₃ represents hydrogen, hydroxyor methoxy, especially hydrogen.

In a further alternative aspect the invention provides compounds offormula (I-f):

wherein:

-   R₁ is hydrogen, alkyl, cycloalkyl or C(O)R₇, and-   R₃ is hydroxy, alkoxy, alkyl, cycloalkyl, halo or OC(O)R₇, with the    exception that R₂ and R₃ are not both hydrogen,-   R₄ is hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, amino,    C₁₋₄-alkylamino or di(C₁₋₄-alkyl)amino, OC(O)R₇ or OR₈, and-   R₇ is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino, and-   R₈ is aryl such as phenyl or arylalkyl such as benzyl.

Preferably in compounds of formula (I-f) R₁ represents hydrogen ormethyl, especially hydrogen.

Preferably in compounds of formula (I-f) R₂ represents hydroxy orC₁₋₆-alkoxy such as methoxy, especially hydroxy.

Preferably in compounds of formula (I-f) R₃ represents hydrogen orC₁₋₆-alkoxy such as methoxy, especially hydrogen.

Preferably in compounds of formula (I-f) R₃ is in the 3-position.

Preferably in compounds of formula (I-f) R_(4a) represents amino,C₁₋₄-alkylamino or di(C₁₋₄-alkyl)amino, especially amino.

Especially preferred compounds of formula (I) include:

-   3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (HMC; Cpd. 1);-   3-(4-hydroxyphenyl)-4-phenylchroman-7-ol (Cpd. 2);-   3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 3);-   3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (Cpd. 4);-   3-(4-hydroxyphenyl)-4-(4-methylphenyl))chroman-7-ol (Cpd. 5);-   3-(4-methoxyphenyl)-4-(4-methoxyphenyl)-7-methoxychroman (Cpd. 6);-   3-(4-hydroxyphenyl)-4-(2,6-dimethoxy-4-hydroxyphenyl)chroman-7-ol    (Cpd. 7);-   3-(4-hydroxyphenyl)-4-(2-hydroxyphenyl)chroman-7-ol (Cpd. 8);-   3-(4-hydroxyphenyl)-4-(3-acyl-2-hydroxy-4-methoxyphenyl)chroman-7-ol    (Cpd. 9);-   3-(3-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 10);-   3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol (HHC; Cpd. 11);-   3-(4-bromophenyl)-4-(4-methoxyphenyl)chroman-7-ol (Cpd. 12);-   3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 13);-   3-(4-hydroxyphenyl)-4-(3-aminophenyl)chroman-7-ol (Cpd. 14);-   3-(4-hydroxyphenyl)-4-(4-phenoxyphenyl)chroman-7-ol (Cpd. 15);-   3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-8-methylchroman-7-ol    (Cpd. 16).

or a pharmaceutically acceptable salt thereof.

The compounds of formula (I) according to the invention include twochiral centres. The present invention includes all the enantiomers anddiastereoisomers as well as mixtures thereof in any proportions. Theinvention also extends to isolated enantiomers or pairs of enantiomers.Methods of separating enantiomers and diastereoisomers are well known toperson skilled in the art.

It will be clear to persons skilled in the art that the in compounds offormula (I) the aryl substituents on the heterocyclic ring can be cis ortrans relative to each other. Preferably in the compounds of formula (I)these substituents will be cis.

A particularly preferred compound of the present invention is thecis-isomer of compound No. (1), HMC:

or a pharmaceutically acceptable salt thereof.

Likewise, particularly preferred compounds are compound Nos. (2) to (16)in the cis-conformation.

The compounds of formulae (III) and (IV) are intermediates as set outherein. Each corresponding isoflavan-4-ol and isoflavan-3-eneintermediate of compound Nos. (1) to (16) are also preferred compoundsof the present invention.

W in compounds of formula (III) and (IV) may, for example, represent thefollowing radicals:

or a protected derivative thereof wherein R₄, R₅ and R₆ are as definedabove for compounds of formula (I).

The term “isoflavone” as used herein is to be taken broadly to includeas isoflavones, isoflavenes, isoflavans, isoflavanones, isoflavanols andthe like.

The term “alkyl” is taken to include straight chain and branched chainsaturated alkyl groups of 1 to 6 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl andthe like. The alkyl group more preferably contains preferably from 1 to4 carbon atoms, especially methyl, ethyl, propyl or isopropyl.

Cycloalkyl includes C₃₋₆ cycloalkyl such as cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

The alkyl group or cycloalkyl group may optionally be substituted by oneor more of fluorine, chlorine, bromine, iodine, carboxyl,C₁-C₄-alkoxycarbonyl, C₁-C₄-alkcylamino-carbonyl,di-(C₁-C₄-alkyl)-amino-carbonyl, hydroxyl, C₁-C₄-alkoxy, formyloxy,C₁-C₄-alkyl-carbonyloxy, C₁-C₄-alkylthio, C₃-C₆-cycloalkyl or phenyl.

Preferably the alkyl group does not bear any substituents.

The term “aryl” is taken to include phenyl, benzyl, biphenyl andnaphthyl and may be optionally substituted by one or more C₁-C₄-alkyl,hydroxy, C₁-C₄-alkoxy, carbonyl, C₁-C₄-alkoxycarbonyl,C₁-C₄-alkylcarbonyloxy, nitro or halo.

The term “halo” is taken to include fluoro, chloro, bromo and iodo,preferably fluoro and chloro, more preferably fluor. Reference to forexample “haloalkyl” will include monohalogenated, dihalogenated and upto perhalogenated alkyl groups. Preferred haloalkyl groups aretrifluommethyl and pentafluoroethyl.

The compounds of the invention include all salts, such as acid additionsalts, anionic salts and zwitterionic salts, and in particular includepharmaceutically acceptable salts as would be known to those skilled inthe art. The term “pharmaceutically acceptable salt” refers to anorganic or inorganic moiety that carries a charge and that can beadministered in association with a pharmaceutical agent, for example, asa counter-cation or counter-anion in a salt. Pharmaceutically acceptablecations are known to those of skilled in the art, and include but arenot limited to sodium, potassium, calcium, zinc and quaternary amine.Pharmaceutically acceptable anions are known to those of skill in theart, and include but are not limited to chloride, acetate, tosylate,citrate, bicarbonate and carbonate.

Pharmaceutically acceptable salts include those formed from: acetic,ascorbic, aspartic, benzoic, benzenesulphonic, citric, cinnamic,ethanesulphonic, fumaric, glutamic, glutaric, gluconic, hydrochloric,hydrobromic, lactic, maleic, malic, methanesulphonic, naphthoic,hydroxynaphthoic, naphthalenesulphonic, naphthalenedisulphonic,naphthaleneacrylic, oleic, oxalic, oxaloacetic, phosphoric, pyruvic,p-toluenesulphonic, tartaric, trifluoroacetic, triphenylacetic,tricarballylic, salicylic, sulphuric, sulphamic, sulphanilic andsuccinic acid.

The term “pharmaceutically acceptable derivative” or “prodrug” refers toa derivative of the active compound that upon administration to therecipient is capable of providing directly or indirectly, the parentcompound or metabolite, or that exhibits activity itself and includesfor example phosphate derivatives and sulphonate derivatives. Thus,derivatives include solvates, pharmaceutically active esters, prodrugsor the like. This also includes derivatives with physiologicallycleavable leaving groups that can be cleaved in vivo to provide thecompounds of the invention or their active moiety. The leaving groupsmay include acyl, phosphate, sulfate, sulfonate, and preferably aremono-, di- and per-acyl oxy-substituted compounds, where one or more ofthe pendant hydroxy groups are protected by an acyl group, preferably anacetyl group. Typically acyloxy substituted compounds of the inventionare readily cleavable to the corresponding hydroxy substitutedcompounds.

Chemical functional group protection, deprotection, synthons and othertechniques known to those skilled in the art may be used whereappropriate to aid in the synthesis of the compounds of the presentinvention, and their starting materials.

The protection of functional groups on the compounds and derivatives ofthe present invention can be carried out by well established methods inthe art, for example as described in T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley & Sons, New York, 1981.

Hydroxyl protecting groups include but are not limited to carboxylicacid esters, eg acetate esters, aryl esters such as benzoate,acetals/ketals such as acetonide and benzylidene, ethers such aso-benzyl and p-methoxy benzyl ether, tetrahydropyranyl ether and silylethers such as t-butyldimethyl silyl ether.

Protecting groups can be removed by, for example, acid or base catalysedhydrolysis or reduction, for example, hydrogenation. Silyl ethers mayrequire hydrogen fluoride or tetrabutylammonium fluoride to be cleaved.

It will be clear to persons skilled in the art of medicinal chemistrythat compounds of formula (I) may be converted into other compounds offormula (I), for example, where a compound of formula (I) bears one ormore hydroxyl substituents then one or more of these substituents can beconverted in to a halo substituent such as bromo, chloro or iodo bytreating the alcohol with a halogenating agent. Halogenating agentsinclude compounds like NBS, hydrobromic acid, chlorine gas etc. It maybe necessary during processes such as halogenation to use protectinggroups to protect other functionality in the molecule.

Phenolic type hydroxyls may not be readily convertible to thecorresponding halogen compound by treatment with a halogenating agent.However, the desired halogen compound may be prepared by, for example,treating an appropriate aryl amine starting material with NaNO₂ in thepresence of HCl under reduced temperature conditions such as 0° C., toform the corresponding azide salt. Subsequent treatment with CuCl, CuBr,KI or HBF₄ may be used to convert the azide into the requiredhalo-compound.

A general process for preparing compounds of formula (I) comprises thestep of treating a compound of formula (IV):

wherein R₁, R₂, R₃ and W are as defined above in relation to compoundsof formula (II) with a reducing agent to provide a compounds of formula(I) or a protected derivative thereof.

Reducing agents are well known to persons skilled in the art and caninclude hydride sources like borohydrides and alkali metal borohydrides,but would include hydrogen in catalytic hydrogenation where a suitablecatalyst such as palladium on carbon may be used. Other suitable hydridesources include sodium triacetoxyborohydride tetrabutyl ammoniumtriacetoxyborohydride and sodium cyanoborohydride.

Preferably the double bond in compounds of formula (IV) is reduced byhydrogenation.

Compounds of formula (IV) are prepared by dehydrating a compound offormula (III):

wherein R₁, R₂, R₃ and W are as defined above, in relation to compoundsof formula (II) or a protected derivative thereof.

Dehydration can, for example, be catalysed by acid, by base orfacilitated by conversion of the tertiary alcohol into a better leavinggroup as would be known to those skilled in the art.

Preferably compounds of formula (III) are dehydrated, for example, bytreatment with para-toluene sulphonic acid.

Compounds of formula (III) may be prepared by treating compounds offormula (II):

wherein R₁, R₂, R₃ are as defined above for compounds of formula (II) ora protected derivative thereof with an arylating agent, for example, acompound of formula W⁻M⁺ wherein W⁻ is an optionally substituted arylradical and M⁺ is one or more counter ions, preferably [MgBr]⁺.

The arylating agent W⁻M⁺ may be prepared by Grignard chemistry where thehaloaryl compound (V):

or a protected derivative thereof wherein

-   R₄, R₅ and R₄ are independently hydrogen, alkoxy, alkyl, acyl,    OC(O)R₇, a protected hydroxy such as OSi(R₁₀)₃ or protected amino    such as trimethylsilylamino phenyl halide, and-   R₁₀ is independently alkyl or aryl, and-   X is halo, preferably bromo,    is reacted with a metal such as magnesium to form the arylating    agent.

Preferably the haloaryl compound (V) is selected from:

wherein R₄, R₅, R₆ and X are as defined above for compounds of formula(V).

Reaction of the arylating agent with the ketone of formula (II) providesaccess to the corresponding isoflavan-4-ols (III), isoflav-3-enes (IV)and isoflavans (I) of the present invention.

Alternatively compounds of formula (III) may be prepared by reactingcompounds of formula (II) with a compound analogous to compounds offormula (V) wherein X represents any appropriate leaving group L whichis lost in the formation of the product by nucleophilic addition of thearyl moiety to a ketone by reactions well known by those skilled in theart.

Preferably any free alcohols, esters or other such reactive groups inthe keto compounds of formula (II) will be protected, for example, ast-butyldimethylsilyl ethers during the nucleophilic addition reaction.

Compounds of formula (II) can be prepared by reducing the eneone doublebond in compounds of formula (VI):

or a protected derivative thereof, wherein R₁, R₂ and R₃ are as definedabove, for compounds of formula (II).

Suitable reducing agents are described above. Preferably reduction ofthe carbon-carbon double bond can be effected, for example, byhydrogenation.

Access to compounds of general formula (VI) is available by generalsynthetic methods as set out in Scheme 1 below and as described inpublished International application No. WO01/17986, the disclosure ofwhich is incorporated herein by reference. A typical synthesis isdepicted in Scheme I.

Access to the variatious 3-phenyl substituted chromans is available byvarying the substitution pattern on the phenylacetic acid derived group.

Access to the 4-phenyl substituted chromans is available by varying thesubstitution pattern of the arylating agent (V).

Analogues of compounds employed in the processes may be used whichinclude a substituent which corresponds to R₉ as defined for compoundsof formula (I).

As used herein, the terms “treatment”, “prophylaxis” or “prevention”,“amelioration” and the like are to be considered in their broadestcontext. In particular, the term “treatment” does not necessarily implythat an animal is treated until total recovery. Accordingly, “treatment”includes amelioration of the symptoms or severity of a particularcondition or preventing or otherwise reducing the risk of developing aparticular condition.

The amount of one or more compounds of formula (I) which is required ina therapeutic treatment according to the invention will depend upon anumber of factors, which include the specific application, the nature ofthe particular compound used, the condition being treated, the mode ofadministration and the condition of the patient.

Compounds of formula (I) may be administered in a manner and amount asis conventionally practised. See, for example, Goodman and Gilman, “Thepharmacological basis of therapeutics”, 7th Edition, (1985). Thespecific dosage utilised will depend upon the condition being treated,the state of the subject, the route of administration and other wellknown factors as indicated above. In general, a daily dose per patientmay be in the range of 0.1 mg to 5 g; typically from 0.5 mg to 1 g;preferably from 50 mg to 200 mg. The length of dosing may range from asingle dose given once every day or two, to twice or thrice daily dosesgiven over the course of from a week to many months to many years asrequired, depending on the severity of the condition to be treated oralleviated.

It will be further understood that for any particular subject, specificdosage regimens should be adjust over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions.

Relatively short-term treatments with the active compounds can be usedto cause stabilisation or shrinkage or remission of cancers. Longer-termtreatments can be employed to prevent the development of cancers inhigh-risk patients.

The production of pharmaceutical compositions for the treatment of thetherapeutic indications herein described are typically prepared byadmixture of the compounds of the invention (for convenience hereafterreferred to as the “active compounds”) with one or more pharmaceuticallyor veterinary acceptable carriers and/or excipients as are well known inthe art.

The carrier must, of course, be acceptable in the sense of beingcompatible with any other ingredients in the formulation and must not bedeleterious to the subject. The carrier or excipient may be a solid or aliquid, or both, and is preferably formulated with the compound as aunit-dose, for example, a tablet, which may contain up to 100% by weightof the active compound, preferably from 0.5% to 59% by weight of theactive compound.

One or more active compounds may be incorporated in the formulations ofthe invention, which may be prepared by any of the well known techniquesof pharmacy consisting essentially of admixing the components,optionally including one or more accessory ingredients. The preferredconcentration of active compound in the drug composition will depend onabsorption, distribution, inactivation, and excretion rates of the drugas well as other factors known to those of skill in the art.

The formulations of the invention include those suitable for oral,rectal, ocular, buccal (for example, sublingual), parenteral (forexample, subcutaneous, intramuscular, intradermal, or intravenous),transdermal administration including mucosal administration via thenose, mouth, vagina or rectum, and as inhalants, although the mostsuitable route in any given case will depend on the nature and severityof the condition being treated and on the nature of the particularactive compound which is being used.

Formulation suitable for oral administration may be presented indiscrete units, such as capsules, sachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above).

In general, the formulations of the invention are prepared by uniformlyand intimately admixing the active compound with a liquid or finelydivided solid carrier, or both, and then, if necessary, shaping theresulting mixture such as to form a unit dosage. For example, a tabletmay be prepared by compressing or moulding a powder or granulescontaining the active compound, optionally with one or more otheringredients.

Compressed tablets may be prepared by compressing, in a suitablemachine, the compound of the free-flowing, such as a powder or granulesoptionally mixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Moulded tablets may be made by moulding, ina suitable machine, the powdered compound moistened with an inert liquidbinder.

Formulations suitable for buccal (sublingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatine and glycerin or sucrose and acacia.

Formulations suitable for ocular administration include liquids, gelsand creams comprising the active compound in an ocularly acceptablecarrier or diluent.

Compositions of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theactive compounds, which preparations are preferably isotonic with theblood of the intended recipient. These preparations are preferablyadministered intravenously, although administration may also be effectedby means of subcutaneous, intramuscular, or intradermal injection. Suchpreparations may conveniently be prepared by admixing the compound withwater or a glycine buffer and rendering the resulting solution sterileand isotonic with the blood. Injectable formulations according to theinvention generally contain from 0.1% to 60% w/v of active compound andcan be administered at a rate of 0.1 ml/minute/kg.

Formulations for infusion, for example, may be prepared employing salineas the carrier and a solubilising agent such as α-cyclodextrin orderivative thereof. Suitable cyclodextrins include α-cyclodextrin,β-cyclodextrin, γ-cyclodextrin, dimethyl-β-cyclodextrin,2-hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-cyclodextrin,3-hydroxypropyl-β-cyclodextrin and tri-methyl-β-cyclodextrin. Morepreferably the cyclodextrin is hydroxypropyl-β-cyclodextrin. Suitablederivatives of cyclodextrins include Captisol® a sulfobutyl etherderivative of cyclodextrin and analogues thereof as described in U.S.Pat. No. 5,134,127.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. Formulations suitable for vaginaladministration are preferably presented as unit dose pessaries. Thesemay be prepared by admixing the active compound with one or moreconventional solid carriers, for example, cocoa butter, and then shapingthe resulting mixture.

Formulations or compositions suitable for topical administration to theskin preferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol, or oil. Carriers which may be used include Vasoline,lanoline, polyethylene glycols, alcohols, and combination of two or morethereof. The active compound is generally present at a concentration offrom 0.1% to 5% w/w, more particularly from 0.5% to 2% w/w. Examples ofsuch compositions include cosmetic skin creams.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 M to 0.2 M concentration with respect tothe said active compound. See for example Brown, L., et al. (1998).

Formulations suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Panchagnula R, et al.,2000) and typically take the form of an optionally buffered aqueoussolution of the active compound: Suitable formulations comprise citrateor Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2M active ingredient.

Formulations suitable for inhalation may be delivered as a spraycomposition in the form of a solution, suspension or emulsion. Theinhalation spray composition may further comprise a pharmaceuticallyacceptable propellant such as carbon dioxide or nitrous oxide or ahydrogen containing fluorocarbon such as 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or mixtures thereof.

The active compounds may be provided in the form of food stuffs, such asbeing added to, admixed into, coated, combined or otherwise added to afood stuff. The term food stuff is used in its widest possible sense andincludes liquid formulations such as drinks including dairy products andother foods, such as health bars, desserts, etc. Food formulationscontaining compounds of the invention can be readily prepared accordingto standard practices.

Therapeutic methods, uses and compositions may be for administration tohumans or other animals, including mammals such as companion anddomestic animals (such as dogs and cats) and livestock animals (such ascattle, sheep, pigs and goats), birds (such as chickens, turkeys,ducks), marine animals including those in the aquaculture setting (suchas fish, crustaceans and shell fish) and the like.

The active compound or pharmaceutically acceptable derivatives prodrugsor salts thereof can also be co-administered with other active materialsthat do not impair the desired action, or with materials that supplementthe desired action, such as antibiotics, antifungals,anti-inflammatories, or antiviral compounds. The active agent cancomprise two or more isoflavones or derivatives thereof in combinationor synergistic mixture. The active compounds can also be administeredwith lipid lowering agents such as probucol and nicotinic acid; plateletaggregation inhibitors such as aspirin; antithrombotic agents such ascoumadin; calcium channel blockers such as verapamil, diltiazem, andnifedipine; angiotensin converting enzyme (ACE) inhibitors such ascaptopril and enalapril, and β-blockers such as propanolol, terbutalol,and labetalol. The compounds can also be administered in combinationwith nonsteriodal anti-inflammatories such as ibuprofen, indomethacin,aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac. Thecompounds can also be administered with corticosteroids or ananti-emetic such as Zofrane®.

Compounds of formula (I) seem to be particularly suitable forco-administration with one or more anti-cancer drugs such as cisplatin,dehydroequol (DHE), taxol (paclitaxel), gemcitabine, doxorubicin,topotecan and/or camptothecin, especially cisplatin, dehydroequol (DHE),taxol. This may result in improved effects in the treatment, for examplein the form of synergistic effects, in comparison to when only one ofthe medicaments is employed. Particularly the compounds of the presentlyclaimed invention, especially HMC (ie compound 1) seem to bechemosensitisers and increase the cytotoxicity of the one or moreanticancer drug co-administered therewith. This seems to be the caseeven though said anticancer drugs work through a variety of differentmechanisms, for example cisplatin is thought to work by interacting withnuclear DNA, taxol is thought to work by blocking cells in the G2/Mphase of the cell cycle and prevent them forming normal mitoticapparatus, gemcitabine is thought to work by incorporating itself intothe DNA of the cell, ultimately preventing mitosis, doxorubicin isthought to be a topoisomerase II inhibitor thereby preventing DNAreplication and transcription and topotecan is thought to be atopoisomerase I inhibitor.

Interestingly, in some situations this increased cytotoxicity tocancerous cells is not associated with a corresponding increase intoxicity to non-cancerous cells.

Whilst this observation has important implications for the treatment ofmany cancers, it is especially important to the treatment of cancerssuch as melanoma, which are extremely difficult to treat.

The co-administration may be simultaneous or sequential. Simultaneousadministration may be effected by the compounds being in the same unitdose, or in individual and discrete unit doses administered at the sameor similar time. Sequential administration may be in any order asrequired and typically will require an ongoing physiological effect ofthe first or initial active agent to be current when the second of lateractive agent is administered, especially where a cumulative orsynergistic effect is desired.

The invention also extends to a pack comprising the combination therapy.

Compounds for use in the preferred synthetic methods of the presentinvention may be derived from any number of sources readily identifiableto a person skilled in the art. For example, daidzein is readilyavailable or can be synthesised by standard methods known in the art.Suitable methods may be found in, for example, published internationalpatent applications WO 98/08503 and WO 00/49009, and references citedtherein, which are incorporated herein in their entirety by reference.

Compounds of the general formulae (II), (III) and (IV) described aboveare intermediates in the production of the active isoflavan compounds offormula (I). These intermediates also represent further aspects of thepresent invention.

Whilst not wishing to be bound by theory the compounds of the presentinvention are thought to regulate a wide variety of signal transductionprocesses within animal cells and that these signal transductionprocesses are involved in a wide range of functions that are vital tothe survival and function of all animal cells. Therefore, thesecompounds have broad-ranging and important health benefits in animalsincluding humans, and in particular have the potential to prevent andtreat important and common human diseases, disorders and functions,which represents a substantial unexpected benefit.

Thus it seems that the compounds of the present invention have activityas TNFα inhibitors. It is hypothesised that TNFα is part of a tightlyregulated cytokine network, activating multiple signal transductionpathways and inducing or suppressing a wide variety of genes. TNFα canprovide a survival signal for cancer cells and hence it has beenreferred to as a tumour promoting factor. As a central mediator ofinflammation, TNFα provides a molecular link between chronicinflammatory stimuli and the subsequent development of malignantdisease. Consequently its inhibition by the compounds of the inventionmay provide one mechanism by which they exert anti-cancer and/oranti-inflammatory activity. Alternatively, these compounds may be usedas chemopreventative agents.

The particular benefits of this invention lie in (a) the large range ofsignal transduction processes targeted by the compounds, (b) the factthat regulation of these various processes includes both up-regulationof some processes and down-regulation of others, and (c) that such abroad and varied effect on signal transduction processes also isaccompanied by an independent effect on a range of important enzymesthat are fundamental to metabolism and steroidogenesis.

The isoflavan compounds of the present invention exhibit good in vitrotoxicity profiles against normal cells. The isoflavans have broadactivity, markedly better than or at least comparable with dehydroequol.The isoflavans are highly active against cancer cells representative ofleukaemia, glioma, prostate, ovarian, breast and lung cancer. Theisoflavan compounds show potent activity against melanoma andcholangiocarcinoma (gall bladder cancer) cell lines. Good activity wasobserved against colorectal cancer cells.

Radio-sensitisation in vivo may be tested for example employing humanepidermoid vulval carcinoma A431 tumours established on the upper legand subjected to several doses of local radiation (to the tumour bearingleg only). A radiation treatment regimen of 2.5 Gy/day for 4 days willdelay tumour growth, and the effect of the radiation dose in combinationwith the test compound could be assessed by monitoring tumour growthdelay. Tumour growth delay of ˜6 days can be expected using radiationalone. Tumour growth delay using orally dosed test compound can bedetermined separately. Evidence of test compound mediatedradio-sensitisation of A431 tumours is then determined by measuringtumour growth delay using a regimen of orally dosed test compoundpre-treated animals followed by the standard radiation therapy regimendescribed above. A mean growth delay of up to 30 days using thecombination treatment compared to up to 10 days using either radiationor test compound monotherapy regimens is evidence of theradio-sensitisation properties of the compounds of the invention.

Radio-sensitisation in vitro may be tested, for example, employingclonogenic assays using human the human epidermoid vulva carcinoma A431cell line to measure response to radiation alone or in combination withtest compounds. A drug dose causing 10% toxicity to the cells may beused in combination with graded doses of radiation. The appropriate doseof compound would be determined by clonogenic assay. Evidence of testcompound mediated radio-sensitisation is shown by, for example, a >20%toxicity to cells using chemoradiation therapy compared to 10% toxicityusing the corresponding monotherapy regimens.

The compounds of the invention are useful in the treatment, preventionor amelioration of diseases associated with aberrant cell survival,aberrant cell proliferation, abnormal cellular migration, abnormalangiogenesis, abnormal estrogen/androgen balance, dysfunctional orabnormal steroid genesis, degeneration including degenerative changeswithin blood vessel walls, inflammation, and immunological imbalance.

The invention is further illustrated by the following non-limitingExamples and accompanying drawings.

EXAMPLES

In the Examples and accompanying drawings which follow the abbreviation“DHE” is used for dehydroequol, “HMC” is used for compound No. 1, being3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-chroman-7-ol and “HHC” is usedfor compound No. 11, being3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)-chroman-7-ol.

1.0. Synthesis Example 1 4′,7-Diacetoxydatdzein

A mixture of daidzein (2.0 g), acetic anhydride (10 ml) and pyridine (2ml) was heated at 105-110 C for 1 h. After cooling the mixture to roomtemperature, it was stirred for a further 30 min during which time thediacetate crystallised from solution. The product was filtered, washedthoroughly with water and recrystallised from methanol to yield4′,7-diacetoxydaidzein as colourless prisms (2.4 g, 90%).

Example 2 7-Acetoxy-3-(4-acetoxyphenyl)chroman-4-one

Palladium-on-charcoal (5%, 0.02 g) was added to a solution of4′,7-diacetoxydaidzein (0.50 g, 1.5 mmol) in ethyl acetate (80 ml) andthe mixture was stirred at room temperature under a hydrogen atmospherefor 72 h. The catalyst was removed by filtration through Celite and theresulting filtrate was evaporated in vacuo. The residue wasrecrystallised from ethanol to yield7-acetoxy-3-(4-acetoxyphenyl)chroman-4-one (0.40 g, 80%) as colourlessplates.

Example 3 7-Hydroxy-3-(4-hydroxyphenyl)chroman-4-one

Imidazole (0.63 g) was added to a suspension of4′,7-diacetoxydihydrodaidzein (0.26 g, 0.08 mmol) in absolute ethanol(5.0 ml) and the mixture was refluxed for 45 min under argon. Thesolution was concentrated under reduced pressure and distilled water (10ml) was added to the residue. The mixture was left overnight underrefrigeration and the resulting precipitate was filtered. The crudeproduct was recrystallised from ethyl acetate/dichloromethane to yield7-hydroxy-3-(4-hydroxyphenyl)chroman-4-one (0.14 g, 71%) as a whitepowder.

Example 47-(tert-Butyldimethlysilyloxy)-3-(4-(tert-butyldimethlysilyloxy)phenyl)chroman-4-one

7-Hydroxy-3-(4-hydroxyphenyl)chroman-4-one 42 g, imidazole 130 g,tert-butyldimethylsilyl chloride 127 g, and N,N-dimethylformamide (500ml) were combined in a 2 L round bottom flask and stirred under nitrogenat room temperature for 16 hours. The reaction was quenched with theaddition of chilled H₂O (200 ml) with the reaction mix cooled in an icebath. The resultant white solid was filtered and rinsed with water.Recrystallisation from ethanol afforded the product as white fluffycrystals (35.7 g).

Example 57-(tert-Butyldimethylsilyloxy)-3-(4-(tert-butyldimethylsilyloxy)phenyl)-4-(4-methoxyphenyl)chroman-4-ol

7-(tert-Butyldimethylsilyloxy)-3-(4-(tert-butyldimethylsilyloxy)phenyl)-4-(4-methoxypenyl)chroman-4-ol25 g was weighed in a 2-neck round bottom flask, and flushed undernitrogen. Anhydrous THF 80 ml was added to the reaction vessel to give aclear slightly yellow solution. A condenser was attached and thereaction vessel placed in an ice bath. Commercial4-methoxyphenylmagnesium bromide (0.5M solution in THF) 225 ml was addedto the reaction mix dropwise over 10 minutes. The reaction was quenchedby the dropwise addition of wet ether (50:50 H₂O:diethyl ether) whilestill under nitrogen, with a white precipitate forming as increasingamounts of H₂O was added. A further amount of H₂O was added to thereaction mix before extraction with diethyl ether.

The organic layers were combined and washed with water, brine, driedover anhydrous MgSO₄ and solvent removed on rotovap to give clear yellowoil which solidified overnight to give an off-white solid. The crudeproduct was used in the next step without purification.

Example 6 3-(4-Hydroxyphenyl)-4-(4-methoxyphenyl)-2H-chromen-7-ol

7-(tert-Butyldimethylsilyloxy)-3-(4-(tert-butyldimethylsilyloxy)phenyl)-4-(4-methoxyphenyl)chroman-4-ol(42 g), pTsOH (435 g), boiling chips and 2.5 L of ethanol were combinedin a 2-neck 5 L round bottom flask with condenser attached. The reactionwas heated at reflux for 3 hours. The solvent was concentrated in vacuoto ˜100 ml before being poured into chilled, stirred water (700 ml). Themixture was then extracted with ethyl acetate, the combined organiclayers washed with water (3×2 L), brine (1×500 ml), dried over anhydrousmagnesium sulphate and filtered and solvent removed in vacuo to givered/brown oil. The oil was dissolved in methanol (˜100 ml) and put infreezer overnight.

A white precipitate had formed overnight, which was filtered off andrinsed with methanol. The filtrate was concentrated in vacuo to give abrown oil.

Example 7 3-(4-Hydroxyphenyl)-4-(4-methoxyphenyl)-chroman-7-ol

3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-2H-chromen-7-ol 25.5 g (70mmoles), 10% Pd/Al₂O₃ 3.95 g and 200 ml of ethanol were combined in a2-neck 500 ml round bottom flask. The reaction was hydrogenated at lowpressure using standard conditions for 3 hours. The reaction wasfiltered through Celite to remove the catalyst, rinsed through withethanol (300 ml). The filtrate was concentrated to ˜50 ml before beingpoured into chilled, stirred water (1.4 L). A pale orange precipitateformed which then formed a brown oil. The mixture was then extractedwith diethyl ether, the combined organic layers washed with water (3×1L), brine (1×500 ml), dried over anhydrous magnesium sulphate andfiltered. The solvent was removed in vacuo to give red/brown oil. Theproduct was recrystallised from diethyl ether (˜100 ml), to give brownsolid which was rinsed with chilled diethyl ether to give off-whitecrystals 11.3 g. The 1H NMR spectrum and numbering scheme being shownbelow.

Peak H δ ppm Shape J Hz integrates Comments C2equatorial 4.14 Dd 10.98 1 C2axial 4.35 Dd 1 Dd is overlapping C3 3.47 Ddd 1 C4 4.20 Dd 5.12 1 C56.71 D 8.05 1 C6 6.36 Dd 2.56, 8.42 1 C8 6.41 D 2.20 1 C9 3.71 S — 3 CT,C6′ 6.61 D — 2 Doublets C3′, CS′ 6.61 D — 2 overlapping for C2″, C6″6.61 D — 2 C2′, C3′, C3″, C5″ 6.61 D — 2 C2″, C3″ Total integration is 8

In the above general methods, the structures may be optionallysubstituted or protected with appropriate substituents, or synthons orderivatives thereof. The compounds may be present as, for example, theirsalts, acetates, benzyl or silyloxy derivatives as can be determined bya skilled synthetic chemist. Hydroxy groups can be readily alkylated(MeI/base), acylated (Ac₂O/Py) or silylated (Cl—SiR₃/base) and likewisedeprotected by standard methods known in the art.

Example 8 3-(4-Hydroxyphenyl)-4-(4-hydroxyphenyl)-chroman-7-ol

3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (3.17 g) wastransferred to a round bottom flask and the flask was purged withnitrogen. 33 wt. % Hydrogen bromide in acetic acid (13 ml) was addeddrop-wise to the flask and the contents were heated to reflux at 130° C.for 7 hours. The reaction mixture was placed in an ice bath and adjustedto pH 6 using sodium hydroxide solution (40% w/v). The mixture wasextracted with ethyl acetate and the ethyl acetate layer was furtherwashed with water and brine prior to drying over magnesium sulphate. Themixture was filtered and the solvent was removed in vacuo to yield abrown solid (2.89 g). The solid was dissolved in minimal ethyl acetateand purified by column chromatography (Silica 60H, 200-400 mesh usingethyl acetate:chloroform (40:60) eluant).3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol was obtained in ˜80%purity and was further purified by semi-preparative high performanceliquid chromatography (HPLC). The ¹H n.m.r. is shown in FIG. 12.

2.0. Materials and Methods 2.1. Tissue Culture

The human pancreatic cancer cell line, HPAC (CRL-2119) was routinelycultured in 1:1 mixture DMEM (Dulbecco's Modified Eagle Medium Sigma)plus Ham's F12 (Sigma) medium containing HEPES (15 mM), insulin (0.002mg/ml), transferrin (0.005 mg/ml), hydrocortisone, (40 ng/ml), epidermalgrowth factor (10 ng/ml). The ovarian cancer cell lines; CP70 wasobtained as a gift from Dr. Gil Mor (Yale University) and cultured in a1:1 mixture DMEM plus Ham's F12 medium, and SKOV-3 (ovarian cancer cellline) was purchased from ATCC and cultured in McCoys 5a medium. Thebreast cancer cell line MDA-MB-468 cultured in Leibovitz's L-15 medium.The melanoma cell line MM200 was obtained as a gift from Peter Hersey(University of Newcastle) and A2058 was obtained as a gift from Dr PeterParsons (QIMR). Both were cultured in DMEM medium.

All cultures were supplemented with 10% FCS (fetal calf serum CSL,Australia), penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine(2 mM) and sodium bicarbonate (1.2 g/L), and cultured at 37° C. in ahumidified atmosphere of 5% CO₂. All cell lines were purchased from ATCC(Maryland, USA) except where noted.

The normal cell line NFF (neonatal foreskin fibroblasts) was a gift fromDr. Peter Parsons (Queensland Institute of Medical Research). RK (rabbitkidney) cells were obtained from Miller Whalley (Macquarie University).Both cell lines were cultured in RPMI supplemented with 10% FCS (CSL,Australia), penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine(2 mM) and sodium bicarbonate (1.2 g/L), and cultured at 37° C. in ahumidified atmosphere of 5% CO₂.

2.2. Proliferation Assays

IC50 values were determined for each cell line. Cells were seeded in96-well plates at an appropriate cell density as determined from growthkinetics analysis and cultured for 5 days in the absence and presence ofthe test compounds. Cell proliferation was assessed after the additionof 20 μl of 3-4,5 dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT,2.5 mg/ml in PBS, Sigma) for 3-4 hrs at 37° C. according tomanufacturer's instructions. IC50 values were calculated from semi-logplots of % of control proliferation on the y-axis against log dose onthe x-axis.

2.3. DHE and HMC Pharmacokinetics—Oral

HMC and DHE were prepared as homogenous suspensions in 1% CMC (m:v,water). Both formulations were delivered orally by savage to femaleBALB/c mice at a dosage of 50 mg/kg. Three animals were allocated toeach timepoint (15 min, 30 min, 1 hr, 4 hr and 24 hr). At eachrespective timepoint, animals were euthanased by cervical dislocationand blood collected. Free HMC was analysed by mass spectroscopy.

2.4. HMC Pharmacokinetics—i.v. and i.p.

HMC was prepared as a solution in 20% hydroxypropyl-beta-cyclodextrin(m:v, water). The formulation was delivered either orally by gavage orby i.p. injection to female BALB/c mice at a dosage of 50 mg/kg. Threeanimals were allocated to each timepoint (15 min, 30 min, 1 hr, 4 hr and24 hr). At each respective timepoint, animals were euthanased bycervical dislocation and blood collected. Urine was also collected andanalysed for HMC. Free HMC was analysed by mass spectroscopy.

2.5 Pilot In Vivo Efficacy Study—HPAC Tumour Bearing Mice

Subconfluent (80%) flasks of HPAC cells were trypsinised, washed inHanks balanced salt solution (Sigma), resuspended in dubellco's minimalessential medium (Sigma) and an equal volume of Matrigel™ (BectonDickson) at a density of 3.7×10⁷ cells per ml. Athymic nu/nu BALB/c micewere s.c. inoculated with 3.7×10⁶ HPAC cells bi-laterally midway alongthe dorsal surface. For the HMC (n=3 per dosage regimen) and controlgroups (n=2), treatment commenced five days post inoculation to allowtumour formation. HMC was formulated 20% HPBCD and delivered i.p. dailyfor 15 days. The control group received equivalent (weight:weight) i.p.doses of 20% HPBCD. Tumour measurements commenced on day 5 postinoculation (10×10 mm²) and were measured in 2 dimensions, length (a)and width (b), using calipers. Tumor weight (W) was calculated by theformula W=ab2/2, where a, is the longer of the 2 measurements (Odwyer etal., 1994). Tumour proliferation curves were analyzed with respect tomaximal tumour inhibition (treated/control, T/C). On sacrifice, liver,kidney, femur, stomach and colon tissue were fixed in buffered formalin,embedded in paraffin, sections cut and stained with H&E. Stainedsections were then submitted to Rothwell consulting for histopathologyanalysis. Serum biochemistry was conducted on bloods taken from control,vehicle control and HMC treatment groups. Serum analysis was conductedby Veterinary Clinical pathology (U. Syd).

2.6 Three-D Model Analysis of Synergy

3-D model analysis of the cytotoxic interaction between drug A and drugB enables the representation of predicted inhibitory effect of two drugsin combination in 3 dimensions to reveal actual regions of synergy orantagonism. The 3D synergy plots are based on a theory of “ThereticalAdditivity” (TA) as outlined by Kanzawa et al (Int. J. Cancer 71,311-319 (1997)). Theoretical Additivity was calculated from thecytotoxicities of drug A and drug B as monotherapies using the followingformula which assumes the drugs are mutually exclusive inhibitors:

${TA}_{(1)} = \frac{{\left( f_{a} \right)A} + \left( f_{a} \right)_{B} - {2\left( f_{a} \right){A\left( f_{a} \right)}_{B}}}{1 - {\left( f_{a} \right)_{A}\left( f_{a\;} \right)_{B}}}$Where: (f_(a))_(A)=fraction of cells affected by drug A

(f_(a))_(B)=fraction of cells affected by drug B

The TA is calculated for each combination of drug concentrations andsubtracted from the observed experimental effect for each combination togive a measurement of synergistic action. A positive differenceindicates that more cells are affected by the drug combination thanwould be expected in theory if the two drugs were administeredtogether—hence synergism. A negative difference indicates that lesscells were affected than theoretically expected—hence antagonism.

3.0. Results 3.1. Normal Cell Toxicity

Dehydroequol (DHE) was less toxic to both NFF and rabbit kidney cellswith IC50 values above 150 μM when compared to HMC (86 and 61 μMrespectively) (Table 1 and FIG. 1). In a separate study, HHC was foundto be non-toxic to both NFF and RK cells (see again Table 1). Whencompared to cisplatin, a benchmark chemotherapeutic agent, the degree oftoxicity exhibited by HMC and HHC is mild.

TABLE 1 Relative toxicity of DHE, HMC, HHC and cisplatin againstNeonatal foreskin fibroblasts (NFF) and rabbit kidney cells.Antineoplastic Tissue/cell Analogue (IC50 μM) (IC50 μM) Type DesignationDHE HMC HNC Cisplatin Fibroblast Neonatal >150 86.12 ± 7.6 60.8 9.85 ± 5Foreskin Fibroblasts (Human, NFF) Kidney Rabbit Kidney >150   61 ±4.3 >150 Not tested

3.2. In Vitro Efficacy Against Cancer Cells

When compared to DHE IC50 values, HMC demonstrated markedly superioractivity (˜5-10 fold greater) against the multi-drug resistant, p53 mtovarian cancer cell line (SKOV-3), the AR negative, p53 Mt prostatecancer cell line (PC3), both ER positive (p53 wt) and negative (p53 mt)breast cancer cell lines (MCF-7 and MDA-MB-468 respectively), p53 MtGlioma (HTB-138), p53 Mt pancreatic cancer (HPAC) and p53 Mt large celllung cancer (Table 2). HMC exhibited anti-cancer activity comparable tothat of DHE against all other cell lines tested. (Table 1). Particularefficacy of HMC was noted against melanoma cells. (Table 2.1 and FIG.2). This represents a substantial advantage over the prior art.

HMC was differentially active against 2 separate colorectal cell lines,with marked activity observed against HT-29 cells and somewhat lessactivity against HCT-15. It is noted that HT-29 and HCT-15 are COX-2positive and deficient respectively. When examined microscopically andcompared to cells treated with vehicle only, HMC treated SKOV-3 cellsexhibited morphological changes consistent with cells undergoingapoptosis (cell enlargement, granular appearance in cytosol andblebbling of plasma membrane). In contrast SKOV-3 cells exposed to 100μM Dehydroequol after 18 hr retained a relatively normal morphology,comparable with that of vehicle only treated cells.

TABLE 2.1 Comparison of Dehydroequol and HMC cytoxicity against celllines representative of different malignancies. Antineoplastic Analogue(IC50 μM) (IC50 μM) Indication Designation DUE HMC Cisplatin OvarianA2780  1.7 ± 0.61 1.58 ± 0.59 2.10 CP70 1.69 ± 0.62 1.21 ± 0.29 10.30SKOV-3 21.83 ± 4.65  2.26 5.40 Prostate PC3 9.09 ± 8.12 2.50 ± 0.92 2.11LNCaP 4.8 ± 3.8 4.52 >10 DU145_(—) 5.95 ± 1.5  3.78 2.07 Breast MCF-721.5 ± 13.2 7.15 ± 7   3.69 MDA-MB- 7.9 ± 3.5  1.1 ± 0.35 0.58 468Glioma HTB-138 7.35 ± 0.89  1.9 ± 0.27 42.30 Pancreatic CRL-2119 56.62 ±16.8  14.1 ± 1.16 9.36 Leukemic RPM1-8226 3.72 ± 0.08 NT NT CCRF-CEM 1.7 ± 0.68 1.90 NT Lung NCI-H23 8.75 ± 7.2  3.75 ± 2.5  NT NCI - H46010.6 ± 3.8  2.23 ± 0.15 22.29 Colorectal HT-29 50.45 ± 21.9  3.7 ± 1.422.7 ± 35   HCT-15  24.4 ± 12.57 37.8 ± 33   129.9 ± 39   Melanoma MM2002.90 0.7 ± .03 8.3 ± 0.7 A2058 NT  1.2 ± 0.65 5.73 ± 2.3  IGR-3 NT 0.53± 0.02 NT

In further studies, the cytotoxicity of various compounds describedherein against various cell lines was determined. Compound 14-ene is the3-ene chromene precursor to the correspondingly reduced chroman,compound 14. It was observed that compounds 1, 2 and 11 show the bestefficacy against most all cancer cell lines. Compound 14 shows slightlybetter efficacy in general compared to its corresponding 14-ene and tocompound 6 (Table 2.2).

TABLE 2.2 Chroman compounds 1, 2, 6, 11 and 14 and chrom-3-ene compound14-ene cytoxicity against cell lines representative of differentmalignancies. Compound (IC50 μM)• Indication Cell line HMC 1 2 6 HHC 1114-ene 14 Ovarian CP70 2.1  3 ± 1.2 >100 1.1 ± 0.9 >100 >100    ProstatePC3 2.5 ± 1   4.2 ± 0.02 116 ± 57 0.88 ± 0.4  46.2 ± 7  32.5 ± 2.1 Breast MDA-MB-468 2.8 ± 4.2 1.9  28.7 ± 3.6  1 ± 0.1 NT 56.6 GliomaHTB-138 1.9 ± 0.3 3.77 >100 0.52 ± 0.1   8.2 ± 10 53.5 ± 14  PancreaticHPAC 2_8 ± 0.9  24.4 ± 12  >100 31.6 ± 27  81.5 ± 59 79 ± 56  LeukaemiaCCRF-CEM   2 ± 0.97 4.02   92 ± 81.6  0.6 ± 0.01 >100  73 ± 51.6 NSCLung NCI-E460 2.8 ± 2  5.4 ± 2.1 NT 0.5 ± 0.1 >100 65   Colorectal HT-295.4 ± 1.8 59.4  >100 2.5 ± 1  97.5 ± 30 45 ± 4.7 Melanoma MM200 1.07 ±0.5  7.34 >100 0.6 ± 0.3  58 ± 2.7 93 ± 2.9

3.3.1. HMC Pharmacokinetics—Oral

When compared with the pharmacokinetic profile of orally dosed DHE, HMCadministered via the same route and dosage (50 mg/kg), HMC exhibited aCmax of 141 μM (achieved after 1 hr) compared to 511 μM for DHE(achieved after 15 min) (Table 3 and FIG. 3). Like DHE, HMC is alsosubject to conjugation with low plasma concentrations of the free formof the molecule observed (1.3 μM after 30 min) (Table 3 and FIG. 3).This is less than half the maximum concentration of free dehydroequolachieved using the same dosage regimen (3.3 μM after 15 min) (FIG. 3).The ratio of free:total is greater for HMC when compared to DHE (0.92 vs0.64 respectively).

TABLE 3.1 Comparison of free and total plasma concentrations achieved inmice dosed with 50 mg/kg of either HMC or DHE p.o. HNC (μM) DHE (μM)Time Total Free Total Free 0.25 72 ± 4.4 0.38 ± 0.04 511.5 ± 99   3.3 ±0.13 0.5 122 ± 18.4 1.3 ± 0.2 357 ± 82 2.9 ± 0.05 1 141 ± 45.8 0.95 ±0.4    387 ± 22.8 1.5 ± 0.11 4 33.9 ± 12.7 0.19 ± 0.08 117.6 ± 42   1.3± 0.07 24 0 0 0.13 ± 0.1 0.15 ± 0.04 

3.3.2. HMC and HHC Pharmacokinetics—Oral

Human patients were orally dosed with 200 mg of either HMC or HHC. Foreach challenged patient, blood was taken over a 6 hour period and theresults averaged to characterise the plasma pharmacokinetics. Theinitial results give an oral half-life of 3.99 hours for HMC and 3.26hours for HHC (Table 3.2).

TABLE 3.2 Comparison of plasma half-life concentrations achieved inhumans dosed with 200 mg of either HMC or HHC Compound C_(max) (ng/mL)T_(max) (h) T1_(1/2) (h) HMC (1) 513 2.17 3.99 HHC (11) 341 2.67 3.26

3.4. HMC Pharmacokinetics—i.v. and i.p.

When formulated in HPBCD and delivered i.v., extremely high levels ofHMC were observed in the blood equating to 1 mM of drug, 15 min postadministration (FIG. 4). Elimination kinetics of i.v. delivered HMC werebiphasic with HMC being rapidly excreted from blood at a rate of 1000μM/hr in the first hr post administration. Assuming linear excretion,this rate slowed to 0.97 μM/hr in hours 1-4 hr post administration. Whenthe same formulation was administered i.p., approximately 1 log less HMCwas observed in plasma (131 μM by i.p. administration vs 1069 μM by i.v.administration) up to 1 hour post administration (FIG. 4). Eliminationkinetics by i.p. administration however, was much slower during thisperiod (112 μM/hr) thus resulting in a serum concentration some 4.5 foldhigher at 1 hr post administration (18.7 by i.p. vs 3.98 by i.v.).Conversely, in hours 1-4 post administration, elimination kinetics wasfaster after i.p. administration when compared to i.v. (4.6 μM/hr byi.p. vs 0.97 μM/hr by i.v.). These data confirm that HMC is highlybioavailable in its free state when administered by i.v. or i.p. routes.In conjunction with oral PK data, these data also suggest that HMC issusceptible to rapid removal by GI detoxification enzymes. Largeconcentrations of free HMC were observed in urine over 0.5, 1 and 4 hrwhere collected (3.3 mM, 3.9 mM and 0.093 mM).

TABLE 4 Comparison of the pharmacokinetic profile of HMC in serum afteri.v and i.p administration of HMC formulated in 20% hydroxypropyl betacyclodextrin at a dose of 50 mg/kg). Inset shows HMC concentrations inserum. Serum HMC (μM) Time (hr) iv ip 0.25 1069.75 131.37 0.50 198.6631.78 1 3.98 18.74 4 0.07 0.15 24 0.05 0.17

3.5. Pilot In Vivo Efficacy Study—HPAC Tumour Bearing Mice

HMC when dosed daily, i.p. at 100 mg/kg significantly retarded theproliferation of HPAC tumours over the treatment period when compared tovehicle control (FIG. 5). When the mean terminal tumour mass wasassessed a significant reduction in final tumour burden (% T/C=62) wasalso noted (FIG. 6). Importantly, no signs of toxicity were noted inanimals dosed with HMC at 100 mg/kg daily for 15 days as determined byweight loss. Indeed animals treated with HMC appeared to thrive whencompared to control (FIG. 7). Organs (liver, kidney, spleen, femur,stomach and colon) were collected and submitted for histopathologicalassessment by Bothwell consulting. A limited serum biochemistry analysiswas also conducted. These data confirm that HMC demonstratesantitumorigenic activity against HPAC tumours in vivo.

3.5.1. Histopathological Examination of HMC Treated Groups

Histopathological examination of haematoxylin and eosin-stained sectionscut from formalin-fixed tissues from two series of experimental mice wasmade. The liver, kidney, stomach and colon were examined for evidence oftoxic damage, the spleen and bone marrow for evidence ofmyelosuppression and the tumour for degree of necrosis. A score of 0-5was allocated to each tumour specimen for the degree of necrosispresent, a 0 score representing, no necrosis and a score of 5, totalnecrosis. The sections were scored ‘blind’ on two separate occasions andthe final score given in the results is the mean of these two scores.

TABLE 5 HMC Toxicology Sample Description Necrosis Score 0-5 4/04 & 1/8Vehicle control 0.5 4/04 & 2/8 HMC 2 4/04 & 4/5 2 4/04 & 5/8 2 4/04 &1/11 No treatment control 0.5

3.5.1.1. Overview of Results

No evidence of toxicity or myelosuppression was detected in sections cutfrom the tissues of the drug-treated mice. However, in all thedrug-treated mice there were patchy mild/moderately severe chronicinflammatory changes affecting the serosa and attached mesentery, aswell as reactive changes of the mesothelial cells, in some of thetissues examined. These changes are consistent with the intra-peritonealinjection of a mildly irritant material.

Significant necrosis of tumour tissue was not detected in controlspecimens 1/8 and 1/11. However, there was considerable necrosis in thetumour sections from the drug-treated mice.

3.5.1.2. Serum Biochemistry of HMC Treated Mice in Comparison to Control

Alakalines phosphatase (ALP), alanine transferase (ALT) and creatine(Cre) were assessed in HMC treated vs control animals. ALP and Crelevels were similar to control and fell within normal ranges (for rat)however, ALT levels in vehicle control and HMC treated groups were muchlower than no treatment control levels.

TABLE 6 Serum biochemistry of HMC treated mice in comparison to controlClinical marker (mice) ALP ALT Cre Urea Sample Group U/L 1.8 L μM mMControl  1/11 U/L 713 7 9.92 Vehicle control 1/8 152 441 26 9.81 HMCtreated 2/8  74 505 17 7.27 (100 mg/kg) 4/8 111 482 8 8.11 5/8 100 494 67.79 Normal ranges) 86-246 84-143 1.5-6 6.3-8* *for rat ALP: Alkalinephosphatase ALT: Alanine aminotransferase Cre: Creatinine

3.6. HMC Induced Apoptosis in Melanoma Cells and Normal Fibroblasts3.6.1. Melanoma

HMC induced apoptosis in all TRAIL-sensitive and -resistant melanomacells at concentrations down to 2 μM (˜7-10% apoptosis) over 24 and 48hrs of exposure (Table 7 and FIG. 8A). At the clinically significantdrug concentration of 4 μM, the incidence of apoptotic cells after 24 hrexposure to HMC rose to 25% and 39% in TRAIL sensitive (MEL-RM) andTRAIL negative (IGR3) cell lines respectively (Table 7 and FIG. 8A). Theincidence of HMC induced apoptosis at 4 μM after 24 hr exposure in theother cell lines was ˜9%. In comparison, the incidence apoptosis inDHE-treated cells at a concentration of 4 μM after 24 hr exposure was0-1%. Over 48 hr at the same concentration of HMC (4 μM), the incidenceof apoptosis rose to 21-42% in all cell lines examined (Table 7 and FIG.8B). DHE was the only other agent to induce moderate levels of apoptosisafter 48 hr exposure at a concentration of 4 μM, but only in ME4405(14%) and Mel-AT (15%) cell lines (Table 7 and FIG. 8B).

TABLE 7 Summary of apoptosis incidence in DHE and HMC treated melanomacells over 24 and 48 hr. Percent Apoptosis Drug TRAIL Trail- Trail +ve/Concen- sensitive resistant Caspase 8 −ve tration MEL-RM ME4405 IGR3Mel-AT (μM) DHE HMC DHE HMC DHE HMC DHE HMC 24 Con- 0 0 0 0 0 0 0 0 hrtrol expo- 1 0 0 0 0 0 8 0 0 sure 2 0 8 2 8 0 11 3 6 4 0 25 2 9 1 39 4 98 2 39 6 11 3 41 8 23 20 5 38 11 19 4 32 10 24 48 Con- 0 0 0 0 0 0 0 0hr trol expo- 1 1 1.5 1 1.5 1 1 1.5 1 sure 2 1.5 8 3 8 1 9 5 7 4 3 22 1442 2 21 15 25 8 10 38 40 20 8 38 43 59 20 12 30 35 18 8 38 40 35

3.6.2. Normal Fibroblasts

Studies were conducted on normal fibroblasts (MRC-5) and TRAIL-sensitivemelanoma cells (ME4405 and MEL-RM) using 8 μM of either DHE or HMC over24 and 48 hr of exposure (FIG. 9). These data demonstrate that HMC, andto a lesser extent DHE, induced marked levels of apoptosis in bothmelanoma cell lines over 24 and 48 hr. Importantly, while promotingprogrammed cell death in malignant cells, normal fibroblasts were shownto both HMC and DHE induced apoptosis at 8 μM drug over 24 and 48 hr ofexposure. These data confirm that HMC is selectively cytotoxic to cancercells.

The isoflavan compounds of the invention exhibit a superior efficacyprofile against all cancers tested when compared to DHE. While HMC ismarginally more toxic than DHE in NFF and RK cells, HMC is markedly lesstoxic than cisplatin. HMC delivered orally in mice is less bioavailablewhen compared to DHE but the ratio of free:total is greater.HPBCD-formulated HMC was markedly bioavailable in its free form whendelivered i.v and i.p. Significant serum concentrations of free HMCpost-delivery i.p. were some 18 fold above that of orally delivered HMC.It has been demonstrated that HMC, formulated in 20% HPBCD and deliveredi.p., exerts a moderate antitumorigenic activity against HPAC tumours invivo. HMC when delivered at 100 mg/kg to mice is not toxic to majororgans as determined by histopathology however, in all the drug-treatedmice there were patchy mild/moderately severe chronic inflammatorychanges affecting the serosa and attached mesentery, as well as reactivechanges of the mesothelial cells which are consistent with theintra-peritoneal injection of a mildly irritant material.

HMC induced moderate-strong levels of apoptosis in TRAIL-resistant andTRAIL-sensitive melanoma cells after both 24 and 48 hrs of exposure.Normal fibroblast cells were resistant to apoptosis after 48 hrsexposure DHE induces mild-moderate levels of apoptosis inTRAIL-resistant and TRAIL-sensitive melanoma cells after 48 hrs ofexposure. Normal fibroblast cells were resistant to apoptosis after 48hr exposure. The ability of both HMC and DHE to induce apoptosis incaspase negative cells suggest that an operational extrinsic programmedcell death pathway is not essential for HMC and DHE mediated apoptosis.

3.7 In Vitro HMC Synergistic Toxicity in Cancer Cells when Combined withCisplatin, Paclitaxel and Gemcitabine, Camptothecin, Topotecan andDoxorubicin 3.7.1. HMC Combination with Cisplatin Against the MM200Melanoma Cell Line

HMC synergy with cisplatin was assessed either in combination over 5days exposure or in sequence (HMC→cisplatin) against the MM200 melanomacell line. It was difficult to assess for synergistic toxicity using achange in IC50 as a measure of synergy due to HMC toxicity asmonotherapy (Table 8). 3D analysis of the data reveals that onlyadditive toxicity was apparent using the 5-day combination protocol(FIG. 11). Evidence of synergy using the HMC-cisplatin combination wasfurther assessed using the HMC→cisplatin sequence (24 hr exposure toeach compound in sequence) against the melanoma cell line MM200. Usingchange in IC50 to assess for synergy it was noted that HMC atconcentrations of 2 μM markedly chemosensitised the MM200 cells tocisplatin by >1000 fold (Table 8). HMC induced chemosensitisation ofMM200 cells to cisplatin was confirmed using 3D analysis of the data(FIG. 11B). These data demonstrate that HMC is able to chemosensitisecancer cells, in this case melanoma, to cisplatin.

TABLE 8 Comparative assessment of synergy between HMC and the cisplatinagainst the Mel-RM melanoma cell line. Average IC50 data for each agentwhen assessed as a monotherapy or in combination are shown TREATMENT(IC50 μM) Change HMC Change DRUG Combined Factor 24 hr first Factorcisplatin 8.82 — 6.32 — — HMC 0.72 — 20.64 — — HMC 10 μM 1.00E−06 HMCeffect — 1.28E−05 >10000 HMC 5 μM 1.00E−06 HMC effect — 2.71E−05 >10000HMC 2 μM 1.00E−06 HMC effect — 1.00E−06 >10000 HMC 1 μM 0.45 HMC effect— 8.29 −1.31

3.7.2 HMC Combination with Gemcitabine Against the Mel-RM Melanoma Cells

HMC synergy with gemcitabine was assessed either in combination over 5days exposure or in sequence (HMC→gemcitabine) against the Mel-RMmelanoma cell line. It was difficult to assess for synergistic toxicityusing a change in IC50 as a measure of synergy due to HMC toxicity asmonotherapy (Table 9). 3D analysis of the data reveals that 5-daycombination protocol did not elicit synergistic toxicity against theMel-RM cell line. Evidence of synergy using the HMC-gemcitabinecombination was further assessed using the HMC→gemcitabine sequence (24hr exposure to each compound in sequence) against the melanoma cell lineMel-RM. Using change in IC50 to assess for synergy it was noted that HMCat concentrations of 2 and 1 μM markedly chemosensitised the Mel-RMcells to gemcitabine by >1000 fold. HMC-induced chemosensitisation ofMel-RM cells to gemcitabine was confirmed using 3D analysis of the data.These data demonstrate that HMC is able to chemosesnsitise cancer cellsto gemcitabine.

TABLE 9 Comparative assessment of synergy between HMC and gemcitabineagainst the Mel-RM melanoma cell line. Average IC50 data for each agentwhen assessed as a monotherapy or in combination are shown. TREATMENT(IC50 μM) Change HMC Change DRUG Combined Factor 24 Hr first Factor HMC0.51 — 27.79 — — HMC 1 μM 1.00E−06 ?HMC effect — 1.76E−03 >1000 HMC 2 μM1.00E−06 ?HMC effect — 1.00E−06 >1000 Gemcitabine 3.88E−03 4.67E−03 — —HMC 0.51 — 27.79 — — HMC 1 μM 1.00E−06 ?HMC effect — 1.00E−06 >1000 HMC2 μM 1.00E−06 ?HMC effect — 3.89E−05  >100

3.7.3. HMC Combination with Paclitaxel Against the 4405 Melanoma CellLine

HMC synergy with paclitaxel was assessed either in combination over 5days exposure or in sequence (HMC→paclitaxel) against the 4405 melanomacell line. It was difficult to assess for synergistic toxicity using achange in IC50 as a measure of synergy due to HMC toxicity asmonotherapy (Table 10). A 30 fold reduction in IC50 was noted in thecombination experiment when compared to the paclitaxel monotherapy.However, 3D analysis of the data revealed that the 5-day combinationprotocol did not elicit synergistic toxicity against the 4405 cell line.Evidence of synergy using the HMC-paclitaxel combination was furtherassessed using the HMC→paclitaxel sequence (24 hr exposure to eachcompound in sequence) against the melanoma cell line 4405. Using changein IC50 to assess for synergy it was noted that HMC at concentrations of2 μM markedly chemosensitised the 4405 cells to paclitaxel by >1000 fold(Table 10). HMC-induced chemosensitisation of MM200 cells to paclitaxelwas confirmed using 3D analysis of the data. These data demonstrate thatHMC is able to chemosensitise cancer cells to paclitaxel.

TABLE 10 Comparative assessment of synergy between HMC and paclitaxelagainst the 4405 melanoma cell line. Average IC50 data for each agentwhen assessed as a monotherapy or in combination are shown. TREATMENT(IC50 μM) Change HMC Change DRUG Combined Factor 24 hr first Factor HMC1 μM 0.006 1.00 — 0.03 −4.44 HMC 2 μM 1.00E−06 5964.400 — 0.01 −1.035Paclitaxel 1.25E−07 — 5.84E−04 — — HMC 1.26  — 55.50 — — HMC 1 μM4.12E−09 30.36 — 5.31E−05 11.00 HMC 2 μM 3.91E−10 ?HMC — 5.48E−09106633.75 effect

3.7.4. HMC Combination with Topotecan Against the MM200 Melanoma CellLine

HMC synergy with topotecan was assessed either in combination over 5days exposure or in sequence (HMC→topotecan) against the MM200 melanomacell line. It was difficult to assess for synergistic toxicity using achange in IC50 as a measure of synergy due to HMC toxicity asmonotherapy (Table 8). 30 analysis of the data confirmed that the 5-daycombination protocol did not elicit synergistic toxicity against theMM200 cell line. Evidence of synergy using the HMC-gemcitabinecombination was further assessed using the HMC→topotecan sequence (24 hrexposure to each compound in sequence) against the melanoma cell lineMM200. Using change in IC50 to assess for synergy it was noted that HMCat a concentration of 2 μM markedly chemosensitised the MM200 cells totopotecan by >1000 fold (Table 11). HMC-induced chemosensitisation ofMM200 cells to topotecan was confirmed using 3D analysis of the data.These data demonstrate that HMC is able to chemosensitise cancer cellsto topotecan. From the 3D analysis the optimum combination of HMC andtopotecan against the MM200 melanoma cell line would appear to be 2 μMHMC and between 1 and 0.1 μM topotecan.

TABLE 11 Comparative assessment of synergy between HMC and topotecanagainst the MM200 melanoma cell line. Average IC50 data for each agentwhen assessed as a monotherapy or in combination are shown. TREATMENT(IC50 μM) Change HMC Change DRUG Combined Factor 24 hr first Factor HMC1 μM 0.115 −14.145 — 0.009 9.304 HMC 2 μM 1.00E−06 ?HMC effect —7.85E−05 >1000 Topotecan 0.095 —  2.216 — — HMC 0.702 — 17.952 — HMC 1μM 0.000 ?HMC effect — 0.044 50.656 HMC 2 μM 1.00E−06 ?HCM effect —1.00E−06 >10000.00

3.7.5. HMC Combination with Camptothecin Against the Mel-RM MelanomaCell Line

HMC synergy with doxorubicin was assessed either in combination over 5days exposure or in sequence (HMC→doxorubicin) against the Mel-RMmelanoma cell line. It was difficult to assess for synergistic toxicityusing a change in IC50 as a measure of synergy due to HMC toxicity asmonotherapy (Table 12). 3D analysis of the data confirmed that the 5-daycombination protocol did not elicit synergistic toxicity against theMel-RM cell line, indeed evidence of antagonism was noted. Evidence ofsynergy using the HMC-doxorubicin combination was further assessed usingthe HMC→doxorubicin sequence protocol (24 hr exposure to each compoundin sequence) against the melanoma cell line Mel-RM. Using change in IC50to assess for synergy it was noted that HMC at a concentration of 2 μMchemosensitised the Mel-RM cells to camptothecin by ˜12 fold (Table 12).3D analysis of the data, however, reveal a marked degree of synergybetween HMC and doxorubicin against Mel-RM cells. These data demonstratethat HMC is able to chemosensitise cancer cells to paclitaxel. From the3D analysis the optimum combination of HMC and camptothecin against theMM200 melanoma cell line would appear to be 2 μM HMC and between 1 and0.1 μM doxorubicin.

TABLE 12 Comparative assessment of synergy between HMC and doxorubicinagainst the Mel-RM melanoma cell line. Average IC50 data for each agentwhen assessed as a monotherapy or in combination are shown. TREATMENT(IC50 μM) Change Change HMC Factor DRUG Combined Factor 24 hr first —Doxorubicin 0.19 — 0.100 — — HMC 0.54 — 50.515 — — HMC 1 μM 0.40 −2.06 —0.062  1.62 HMC 2 μM 0.18 ?HMC effect — 8.16E−03 12.22

3.8 Inhibition of TNFα in Murine Macrophages (RAW 264.7) by Compounds 4,6 & 7

The mouse macrophage cell line RAW 264.7 was cultured in DMEMsupplemented with FCS, 2 mM glutamine and 50 U/mlpenicillin/streptomycin. Subconfluent cells were detached from the flaskby gentle scraping and 24-well plates seeded at 5×10⁵ cells per well andallowed to adhere for 1 hr. Cells were treated with either test agent(in 0.025% DMSO) or vehicle alone, 1 hr prior to the addition of 50ng/ml LPS. After incubation for 16 hrs, culture media was collected andstored at −80° C. for TNFα. measurement using an enzyme immunometricassay (Becton Dickinson).

Compound 6 and compound 7 of the present invention were tested and theresults are shown in FIG. 12, which indicated that the compounds testedinhibit TNFα. in murine macrophages in a dose dependent manner over theconcentration ranges tested.

The raw data for compounds 6, 7 and additionally compound 4 is shown inTable 13 below.

TABLE 13 Inhibition of TNFα in murine macrophages by compounds 4, 6 and7 Concentration (μM) Compound 6 Compound 7 Compound 4 10 −48.7 −26.2−6.1 1 −13.0 −25.4 −11.9 0.1 −10.4 −1.4 −6.3 0.001 1.0 19.2 −14.3 0.01 —— −11.3

3.9 Assessment of HHC as a Chemosensitiser

HHC was screened as chemosensitiser against a panel cell linesrepresentative of a range cancer indications using a panel of cytotoxicscommonly used in the treatment of cancer. It has emerged that HHC has anability to strongly chemosensitise cancer cell lines from differentpathologies to gemcitabine (ovarian, prostate, breast and pancreaticcancers, and glioma) (Table 14). Strong synergy has been noted usingHHC:cisplatin against ovarian and prostate cancer, mild synergy againstcolorectal cancer cell lines and synergy was not observed in pancreaticcancer and glioma. Moderate synergy has been noted using theHHC:paclitaxel combination against breast and colorectal cancer, andmelanoma cell lines. Equivocal synergy data using the HHC:paclitaxelcombination has been noted against ovarian cancer and glioma cell linesand there was no evidence of synergy against prostate and pancreaticcancer cell lines. Data has revealed that HHC is able to stronglychemosensitise the MM96L melanoma cell line to cisplatin, carboplatinand decarbazine (Table 7).

TABLE 14 Assessment of HHC as a chemosensitiser using a panel of cancercell lines and standard cytotoxics Cancer Indication Ovar- Pros- BreastMela- Glioma Pancre- ian tate MDA- noma HTB- atic Drug CP70 PCS 468MM96L 138 HPAC Cisplatin SSSSS SSSSS — SSSSS n.o. n.o. — SSSSS — — n.o.— — — — — Gemcitabine SSSSS SSSSS SSSSS — SSSSS SSSSS — — — — SSSSS —Paclitaxel n.o. n.o. MS — MS n.o. MS — — — n.o. — — — — — n.o. —Carboplatin — .. — SSSSS — — Dacarbazine — — — SSSSS — — Key: SSSSS =Synergy MS = moderate synergy n.o. = not observed * = not tested

3.10. Efficacy of DHE, HMC and HHC Against Selected Melanoma Cell Lines

In comparison with DHE, both HMC and HHC showed excellent anti-canceractivity against a range of melanoma cell lines. HHC was the mostefficacious agent against all melanoma cell lines tested to date havingsub 1 μM IC50 values (Table 15).

The invention has been described herein, with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation.

TABLE 15 Comparison of DHE, HMC and HHC efficacy against melanomamonotherapy Melanoma coining (IC58 μM) Analogue MM200 A2058 IgR3 RM 4405MM96L SKMe128 DHE 4.77 ± 4   NT NT NT NT 4.76 4.40 HMC 1.13 ± 0.52 1.42± 0.48 0.51 ± 0.08 1.4 ± 1.6 1.46 ± 0.18 1.31 ± 0.38 0.68 ± 0.16 HHC0.66 ± 0.28 0.34 0.20 0.39 ± 0.28 0.50  0.2 ± 0.09 0.36 ± 0.34

4.0 Effect on Murine Macrophages (RAW 264.7) Stimulated with LPS

The mouse macrophage cell line RAW 264.7 was cultured in DMEMsupplemented with foetal calf serum (FCS), 2 mM glutamine and 50 U/mlpenicillin/streptomycin. Subconfluent cells were detached from the flaskby gentle scraping and 24-well plates seeded at 5×10⁵ cells per well andallowed to adhere for 1 hr. Cells were then treated either test compoundat a concentration of 10 μM (in 0.025% DMSO) or vehicle alone, andincubated for 1 hr. LPS 50 ng/ml (LPS-Sigma-Aldrich) was then added.After incubation for 16 hrs, culture media was collected and stored at−80° C. for ecosanoid measurements using enzyme immunometric assays(PGE₂ and TXB₂—Cayman Chemical).

TABLE 16 Percentage change in eicosanoid synthesis after incubating testcompound at 10 μM compared with incubation with vehicle alone. Positivevalues indicate enhanced synthesis; negative values indicate inhibitionof synthesis and consequently suggest anti-inflammatory activity.Compound PGE₂ TXB₂ 1 −33.8 0 2 −12.6 16 6 −37.7 −16.4 11 27.2 51.4

The invention has been described herein, with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. However, a person havingordinary skill in the art will readily recognise that many of thecomponents and parameters may be varied or modified to a certain extentwithout departing from the scope of the invention. Furthermore, titles,headings, or the like are provided to enhance the reader's comprehensionof this document, and should not be read as limiting the scope of thepresent invention.

The entire disclosures of all applications, patents and publications,cited herein, if any, are hereby incorporated by reference.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification individually or collectively, andany and all combinations of any two or more of said steps or features.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in the field ofendeavour.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

SELECTED REFERENCE ARTICLES

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What is claimed is:
 1. A method of treating cancer, the method comprising administering to a subject in need thereof a compound of the formula (I-a):

wherein R₁ is alkyl; R₂ and R₃ are independently hydrogen, hydroxy, alkoxy, halo, or alkyl, with the exception that R₂ and R₃ are not both hydrogen; and R₄, R₅ and R₆ are independently hydrogen, hydroxy, alkoxy, or alkyl; or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein the cancer is of epithelial, mesenchymal, or neural origin.
 3. The method of claim 2, wherein the cancer is ovarian cancer, breast cancer, pancreatic cancer, or melanoma.
 4. The method of claim 1, wherein administering the compound of formula (I-a) results in the cancer being sensitized to one or more chemotherapeutic agent or radiotherapy, and the method further comprises administering to the subject a chemotherapeutic agent or radiotherapy to which the cancer has been sensitized.
 5. The method of claim 4, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, paclitaxel, gemcitabine, doxorubicin, decarbazine, or topotecan.
 6. A method of sensitizing a cancer cell, the method comprising contacting the cancer cell with a compound of formula (I-a):

wherein R₁ is alkyl; R₂ and R₃ are independently hydrogen, hydroxy, alkoxy, halo or alkyl, with the exception that R₂ and R₃ are not both hydrogen; and R₄, R₅ and R₆ are independently hydrogen, hydroxy, alkoxy, or alkyl; or a pharmaceutically acceptable salt thereof.
 7. The method of claim 6, wherein the cancer is of epithelial, mesenchymal, or neural origin.
 8. The method of claim 7, wherein the cancer is ovarian cancer, breast cancer, pancreatic cancer, or melanoma.
 9. The method of claim of 6, wherein the method of sensitizing comprises sensitizing the cancer cell to a chemotherapeutic agent to which, prior to the method of sensitizing, the cancer cell is not sensitive or is poorly sensitive.
 10. The method of claim 6, wherein the method of sensitizing comprises sensitizing the cancer cell to radiotherapy to which, prior to the method of sensitizing, the cancer cell is not sensitive or is poorly sensitive. 